ABSTRACT Real-time fluorescence intravital microscopy has provided compelling evidence that platelet-neutrophil interactions on the activated endothelium are a major determinant of microvascular occlusion during vascular inflammation. Previous studies demonstrated that neutrophil Mac-1 (?M?2 integrin) plays a critical role in platelet-neutrophil interactions under inflammatory conditions. Further, we found that neutrophil AKT2 is a critical regulator for the membrane translocation and activation of Mac-1 during cell activation. However, the detailed mechanisms by which AKT2 regulates Mac-1 function remain unclear. Our preliminary studies showed that neutrophil AKT2 phosphorylates stromal interaction molecule 1 (STIM1), a Ca2+ sensor essential for store- operated Ca2+ entry (SOCE). Importantly, peptides mimicking AKT2 phosphorylation sites on STIM1 significantly inhibited SOCE during neutrophil activation, suggesting the important role of AKT2-phosphorylated STIM1 in inducing SOCE. Another key result is that neutrophil NADPH oxidase 2 (NOX2) is important for mediating Mac- 1 activation but not membrane translocation. In this proposal, we will test the novel hypothesis that neutrophil AKT2-induced SOCE stimulates reactive oxygen species (ROS) generation and ligand-binding activity of Mac- 1, thereby promoting the deleterious neutrophil-platelet interaction and microvascular occlusion under inflammatory conditions. We propose the following specific aims to test this hypothesis: AIM 1: Determine the role of neutrophil AKT2 in stimulating STIM1 and Mac-1 function. We will define the role of AKT2 in mediating SOCE through STIM1 phosphorylation. We will also address how AKT2-induced SOCE stimulates ligand-binding activity of Mac-1. AIM 2: Define the role of neutrophil AKT2-induced SOCE in activating NOX2 and thiol-disulfide exposure in Mac-1. We will determine the role of AKT2-induced SOCE in activating NOX2 and investigate whether AKT2-NOX2 signaling induces thiol exchange in the extracellular domain of Mac-1 and stimulates its ligand-binding function through cell surface PDI activity. We will also identify Cys residues in allosteric disulfide bonds of Mac-1. AIM 3: Investigate the pathophysiologic role of AKT2-induced SOCE in mediating neutrophil-platelet interaction and microvascular occlusion as a central mechanism of vascular inflammation. We will determine the role of AKT2-induced SOCE in neutrophil recruitment, neutrophil-platelet interactions, and vascular occlusion under inflammatory conditions. Further, we will also examine the role of AKT2-induced SOCE signaling in heterotypic cell-cell interaction and aggregation during vaso-occlusive events in sickle cell disease (SCD) patients and mice.